Rapid molecular methods for identification of pathogens and characterization of resistance mechanisms are essential to clinical infectious disease diagnostics. Our work aims to develop novel applications of mass spectrometry to these problems in the clinical microbiology laboratory. Our approach involves both the identification of new bacterial protein markers and the development of assays to detect these markers using LC-MS/MS and other mass spectrometry technologies. Methods are being pursued for (1) Culture-free identification of pathogenic bacteria in primary specimens, (2) Strain-level classification of bacterial pathogens, and (3) Rapid resistance protein identification. Previously published work involved identification of a peptide marker for tracking a carbapenemase-carrying resistance plasmid that could be detected by MALDI-TOF mass spectrometry (Lau et al, JCM, 2014), followed by a clinical validation study demonstrating the method's utility in identifying KPC carbapenemase proteins in clinical isolates from NIH Clinical Center patients (Youn et al, JCM, 2016). Work done during the 2016 fiscal year in collaboration with Anthony Suffredini's group in CCMD, NIHCC included the development of a genoproteomics approach for identifying strain-specific peptide markers based on LC-MS/MS profiling of tryptic peptides, using clinical Acinetobacter baumannii isolates as a model (Wang et al, Clinical Chemistry, 2016). This approach is based on in silico computational analysis to guide selection of informative, genome-specific tryptic peptides from LC-MS/MS peptide profiles, followed by experimental confirmation. Work completed during the past fiscal year included extension of genoproteomic methods to the design of assays for the culture-free identification of bacteria in primary specimens, published in Wang et al, Clinical Chemistry, 2017. This proof-of-concept study demonstrated that rapid (90 minute) diagnostic identification of bacterial pathogens in primary specimens may be possible for a variety of specimen types without culture. The genoproteomic approach was also extended to the direct identification of the KPC carbapenemase in clinical isolates using specific tryptic peptides that can be detected by LC-MS/MS, published in Wang et al, Scientific Reports, 2017. This method allows a rapid (90 minute) diagnostic mass spectrometry assay for detecting the KPC carbapenemase protein in clinical isolates. Work is currently underway to extend culture free identification methods to a variety of high impact pathogenic bacteria. Additionally, work is underway to develop LC-MS/MS assays to detect other carbapenemases and extended spectrum beta lactamses using tryptic peptides and LC-MS/MS, which may be used in multiplex.